OBJECTIVETo investigate the influence of tissue inhibitor of metalloproteinase 2 (TIMP-2) on the infiltrative patterns of human monocytic leukemic cell line SHI-1 in nude mice.

METHODS1) 1 x 10(7) TIMP-2 gene transduced SHI-1 (SHI-1-TIMP-2) and SHI-1 transduced MSCV gene (SHI-1-MSCV) cells were inoculated via tail vein into 6-week nude mice, which pretreated by splenectomy, cytoxan intraperitoneal injection, and sublethal irradiation(referred as SCI nude mice). 30 days after inoculation, half of the mice were sacrificed, and the infiltration patterns were investigated by histological exam and human CD45 immunohistochemistry, other mice were observed for survival time. 2) Leukemic cells inoculated subcutaneously into the axillary area of mice without any pre-treatment. On day 23 and 30, mice were sacrificed to measure the volume of neoplasm. TIMP-2 protein expression and the micro vein density were detected by immunohistochemistry.

RESULTSIn SCI nude mice inoculated via caudal vein with SHI-1-TIMP-2 cells, the survival time was shorter and infiltration (including in central nervous system) was higher than that in those inoculated with SHI-1-MSCV cells. However, in inoculated subcutaneously group, the neoplasm though grew rapidly at first, over expression of TIMP-2 limited the tumor growth and angiogenesis.

CONCLUSIONThe functions of TIMP-2 are diversity; the role of TIMP-2 in tumor infiltration and metastasis was worthy of further investigation.

Animals ; Cell Line, Tumor ; DNA, Complementary ; genetics ; Genetic Vectors ; Humans ; Leukemia, Experimental ; genetics ; pathology ; Leukemic Infiltration ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Tissue Inhibitor of Metalloproteinase-2 ; genetics ; Transfection

Objective Sperm screening is an essential step in IVF procedures. The swim-up method, an assay on sperm motility, is used clinically to select the ideal sperm for subsequent manipulation. However, additional parameters, including acrosome reaction capability, chemotaxis, and thermotaxis are also important indicators of mammalian sperm health. To monitor both sperm motility and chemotaxis simultaneously during sperm screening, we designed and constructed a microdevice comprising a straight channel connected with a bi-branch channel that mimics the mammalian female reproductive tract. Methods The width and length of the straight channel were optimized to select the motile sperm. Cumulus cells were selectively cultured in the bi-branch channel to generate a chemoattractant-forming chemical gradient. Sperm chemotaxis was represented by the ratio of the sperm swimming towards different branches. Results The percentage of motile sperm was improved from ( 58. 5 ± 3. 8 ) % to ( 82. 6±2.9)% by a straight channel 7 mm in length and 1 mm in width. About 10% of sperm were found chemotactically responsive in our experiment, which is consistent with previous studies. Conclusion The combined evaluation of both sperm motility and chemotaxis was achieved for the first time, and the motile and chemotactically responsive sperm can be easily enriched on a lab-on-a-chip device to improve IVF outcome.

BACKGROUNDInteractions of tumor cells with the microenvironment were deemed to promote the tumor invasion and metastasis. CXC chemokine receptor 4 (CXCR4) and extracellular matrix metalloproteinase inducer (EMMPRIN) had reported to participate in this process. However the roles of bone marrow microenvironment in leukemic infiltration were not well investigated.

METHODSA co-culture system between SHI-1 cells and bone marrow stromal cells (BMSCs) is used to simulate the interactions of leukemic cells with their microenvironment. The trans-matrigel invasion was used to detect the capability of SHI-1 cells invasion. The BMSCs and SHI-1 cells were mixed in a ratio of 1:10 and added to the millicell chamber coated with matrigel. Either the co-culture supernatant or the functional blocking peptide of CXCR4 and EMMPRIN were added to the trans-matrigel invasion system. The expressions of EMMPRIN in SHI-1 cells and BMSCs were detected by RT-PCR. The changes of the expression of matrix metalloproteinase-2, 9 (MMP-2, MMP-9), tissue inhibitor of metalloproteinase 2 (TIMP-2), and CXCR4 mRNA in SHI-1 cells were determined by real-time PCR. The concentration of stromal cell derived factor 1 (SDF-1) in serum free supernatant was measured by ELISA.

RESULTSBoth SHI-1 cells and BMSCs express EMMPRIN. SHI-1 cells could hardly invade the matrigel membrane; the coculture supernatant did not induce the invasion of SHI-1 cells. When contacting directly with BMSCs, SHI-1 cells invaded to the lower chamber of millicell were significantly increased. The functional blocking peptide of CXCR4 and EMMPRIN could significantly inhibit the invasion triggered by BMSCs. When co-culturing with BMSCs, the expression of CXCR4, MMP-2, MMP-9 and TIMP-2 mRNA in SHI-1 cells were significantly elevated in company with a significantly higher level of SDF-1 in the co-cultured serum-free supernatant.

CONCLUSIONThe interactions of leukemic cells and BMSCs play important roles in leukemic cell infiltration.

Basigin ; physiology ; Cell Communication ; Cell Line, Tumor ; Coculture Techniques ; Humans ; Leukemia, Monocytic, Acute ; pathology ; Mesenchymal Stromal Cells ; physiology ; Neoplasm Invasiveness ; Receptors, CXCR4 ; physiology

OBJECTIVETo evaluate the prevalence and distribution of C-kit, NPM1 and FLT3 gene mutations in patients with acute myeloid leukemia (AML), and to analyze the relationship between the gene mutations and their prognosis.

METHODSMutations in exon 8 and 17 of C-kit gene, exon 12 of NPM1 gene, exon 20 of FLT3-TKD gene, and exon 14/15 of FLT3-ITD gene were detected by direct sequencing. Clinical data was collected and followed up if the patient had accepted treatment in our hospital.

RESULTSAmong the 656 AML patients, mutations in C-kit exon 8 were found in 6 patients (0.9%), C-kit exon 17 in 33 (5.0%), NPM1 in 169 (25.8%), FLT3-TKD in 46 (7.1%), and FLT3-ITD in 178 (27.1%). Six subtypes of mutations were detected in C-kit exon 8, 8 in C-kit exon 17, 11 in FLT3-TKD, 15 in NPM1, of which 5 were not reported before. C-kit exon 17 mutations were more frequently detected in patients with t(8;21) and exon 8 in patients with inv(16) cytogenetic abnormality. No other gene mutations except FLT3 were detected in M(3) patients. NPM1 and ITD mutations were often detected in individuals with normal cytogenetics or M(5) and M(1) of FAB classification, and accompanied with high white blood cell counts in peripheral blood, high blast counts in bone marrow and low CD34 expression. The older the patients were when diagnosed, the more gene mutations and the higher white blood cell count were detected. More mutations were found in individuals with normal karyotype than that with other karyotypes. It appeared that FLT3-ITD was significantly associated with shorter overall survival (OS) (P = 0.004), NPM1 was not significantly associated with OS, but NPM1(+)/ITD(-) patients had the longest OS.

CONCLUSIONSOur results showed that the mutation types and amounts had particular distribution in MICM subtypes, and were associated with white blood cell counts in peripheral blood, blast counts in bone marrow and prognosis. Especially for patients with normal karyotype, the genetic mutations could be new molecule marker.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Asian Continental Ancestry Group ; genetics ; DNA Mutational Analysis ; Female ; Humans ; Karyotyping ; Leukemia, Myeloid, Acute ; diagnosis ; genetics ; Male ; Middle Aged ; Mutation ; Nuclear Proteins ; genetics ; Prognosis ; Proto-Oncogene Proteins c-kit ; genetics ; Young Adult ; fms-Like Tyrosine Kinase 3 ; genetics

OBJECTIVETo establish a real-time quantitative reverse transcriptase polymerase chain reaction (RQ-RT-PCR) for quantitative detection of the common molecular markers that have affirmative clinical significance in the acute and chronic leukemia patients, and evaluate its significance in diagnosing leukemias and monitoring minimal residual disease (MRD).

METHODSPrimers and TaqMan probes were designed for detecting various fusion transcripts and normal abl gene was used as the internal control. The expression level of fusion transcripts in 202 newly diagnosed leukemias were determined.

RESULTSIn absolute quantity, expression level of the fusion transcripts in various leukemias was b3a2 (b2a2) 47614.63, e1a2 98847.53, AML1-ETO 300029.51, PML-RAR alpha 25506.28, respectively, while in relative quantity to abl, the levels were 1.05, 0.91, 5.33 and 0.55, respectively.

CONCLUSIONThe relative quantification of gene expression level by using RQ-RT-PCR to abl control gene is more accurate and direct viewing. Different levels of transcription of corresponding fusion genes are found in various subtypes of leukemias at diagnosis, among which the level of AML1-ETO was higher and PML-RAR alpha lower.

Biomarkers, Tumor ; genetics ; metabolism ; Core Binding Factor Alpha 2 Subunit ; genetics ; metabolism ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Humans ; Leukemia ; diagnosis ; genetics ; metabolism ; Oncogene Proteins, Fusion ; genetics ; metabolism ; RUNX1 Translocation Partner 1 Protein ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription, Genetic

OBJECTIVETo study the relationship between pretransplantation host thymic recent output function and prognosis in HLA-matched sibling bone marrow transplantation (MSD-BMT) and determine whether pretransplantation host thymic recent output function can act as a marker for predication of prognosis after HSCT.

METHODST-cell receptor excision circle (TREC) in DNA of pretransplantation peripheral blood mononuclear cells from 64 patients underwent MSD-BMT was detected by real-time quantitative PCR. The content of TREC in 70 normal donors was detected as well. All clinical data of patients after HSCT were collected and studied. Survival rates of patients after HSCT were estimated with Log-rank test. Univariate and multivariate analysis of prognostic factors were carried out by COX's proportional hazard regression model.

RESULTSThe mean value of TREC in normal donors was (3351 +/- 3711) copies/10(5) cells. There was an inverse correlation between TREC and age in the donor groups. Before transplantation, all patients were detected TREC, with a mean TREC number of (180 +/-332) copies/10(5) cells being significantly lower than that of normal donors. The results of univariate analysis showed that the counts of pre-HSCT TREC were closely, correlated with long term survival and chronic graft versus host disease (cGVHD) (P < 0.05) and with CMV infection (P = 0.084) but not with acute graft versus host disease (aGVHD). The results of multivariate analysis showed the same thing as that of univariate analysis.

CONCLUSIONPretransplantation host thymic recent output function is closely correlated with prognosis in MSD-BMT and can be a factor for predicting the outcome of HSCT.

Adolescent ; Adult ; Child ; Female ; Hematopoietic Stem Cell Transplantation ; methods ; Humans ; Male ; Middle Aged ; Prognosis ; Receptors, Antigen, T-Cell ; genetics ; Retrospective Studies ; Siblings ; Thymus Gland ; immunology ; Transplantation, Homologous ; immunology ; methods

This study was aimed to investigate the biological characteristics of osteoblasts and their hematopoietic supportive function by using human fetal osteoblastic cell line 1.19 (hFOBs) as a model. The pluripotency markers (Oct-4, Rex-1, hTERT) of hFOBs were analyzed by RT-PCR, the multilineage differentiation experiments were conducted in vitro. Flow cytometry (FCM) was used to identify the surface markers of hFOBs, and RT-PCR was used to analyze their hematopoietic cytokine expression in comparison with bone marrow mesenchymal stem cell (BM-MSC). The results showed that hFOBs expressed several ESC pluripotency markers including Oct-4 and Rex-1, except hTERT. Moreover, hFOBs could also undergo multilineage differentiation into the mesodermal lineages of adipocytic cell types in addition to its predetermined pathway, the mature osteoblast. Both hFOBs and BM-MSC expressed CD44, CD73 (SH3), CD105 (SH2) and CD90 (Thy1), and lack expression of CD34, CD45, or HLA-DR surface molecules. In addition, both hFOBs and BM-MSC expressed SCF, IL-6, and SDF-1alpha mRNA, but only hFOBs could express GM-CSF and G-CSF. It is concluded that human fetal osteoblastic cell line 1.19 may provide a good model to study the osteoblastic regulation role in hematopoiesis in vitro.
Cell Differentiation ; physiology ; Cell Line ; Fetus ; Hematopoiesis ; physiology ; Humans ; Mesenchymal Stromal Cells ; cytology ; physiology ; Models, Biological ; Osteoblasts ; cytology ; physiology

This study was aimed to investigate the expressions of human telomerase reverse transcriptase (hTERT) and survivin gene in patients with myelodysplastic syndrome (MDS), and to explore their relationship. The expression of hTERT mRNA in bone marrow mononuclear cells (BMMNCs) of 56 patients with MDS and 27 patients with iron deficiency anemia were detected by RT-PCR, the expressions of survivin gene in BMMNCs of 55 patients with MDS and 12 patients with iron deficiency anemia were detected by real-time RT-PCR. The results showed that the expression of hTERT significantly elevated in RA and RAEB patients, as compared with controls (p<0.005). With the disease alleviated, the expression of hTERT decreased and had no significant difference from the controls (p>0.25). There was no significant difference in expression of hTERT between low+int-1 risk group and int-2+high-risk group by IPSS (p>0.50). The expression of survivin gene significantly increased in RA and RAEB patients, as compared with controls (p<0.02, p<0.05). The expression of survivin gene in low+int-1 risk group by IPSS was significantly higher than that in the controls (p<0.02), and there was no significant difference in expression of survivin gene between int-2+high-risk group patients and the controls (p>0.10). It is concluded that the expressions of hTERT and survivin may play a critical role in escaping malignant clone of MDS from apoptosis and acquiring the ability to divide unlimitedly.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Apoptosis ; genetics ; Female ; Humans ; Inhibitor of Apoptosis Proteins ; Male ; Microtubule-Associated Proteins ; genetics ; metabolism ; Middle Aged ; Myelodysplastic Syndromes ; enzymology ; genetics ; RNA, Messenger ; genetics ; metabolism ; Telomerase ; genetics ; metabolism ; Young Adult

OBJECTIVETo find out the distributed characteristics of KIR2DL1 alleles frequencies and the recognition HLA-C ligand in the Chinese Han population.

METHODSThe 111 patients and 116 donors from CMDP were performed the KIR2DL1 high-resolution typing and KIR genotyping using sequence-based testing (SBT) and PCR-SSP methods.

RESULTSA total of 224 individuals with KIR2DL1 locus was predominantly observed and accounted for 98.68% (224/227). There were 3 different KIR2DL1 alleles, including KIR2DL1*00302, *00201 and *00401 alleles polymorphism. The most common phenotype observed were KIR2DL1*00302 (84.82%, 380/448), KIR2DL1*00201 (12.05%, 54/448) and KIR2DL1*00401(3.13%,14/448), present at allele genotype frequencies of 61.04%,6.22% and 1.58% respectively. The allele homozygotic types of KIR2DL1*00302 and KIR2DL1*00302 were the most frequent in 6 KIR2DL1 allele by high resolution typing. The allele heterozygous types of KIR2DL1*00302 and KIR2DL1*00401 presented statistically different in haplotypes A/A and B/x (P=0.001), and KIR2DL1*00401 lacked of all A/A haplotype. The KIR2DL1*00302 and KIR2DL1*00201 allele had significant positive associations between different KIR pairs of KIR2DS1, KIR2DL3, KIR2DS4 and KIR3DL1/S1 using linkage disequilibrium analysis (P<0.01), respectively. In the receptor-ligand of KIR/HLA model after allo-HSCT, KIR2DL1*00302 alleles correlated with their HLA-C2 group ligands. KIR2DL1*00302 and HLA-C*06:02 was the most common combination ligand model, but KIR2DL1*00302 and HLA-C*01:02 was the most frequent mismatch ligand model with the development of NK cell-induced alloreactivity, meanwhile there was statistically significant difference of frequency distribution (P<0.05).

CONCLUSIONThe KIR2DL1*00302 was the most frequent allele in Chinese Han population. The KIR2DL1 high resolution typing would be beneficial for predicting donor NK cells all activity after hematopoietic stem cell transplantation and selecting suitable donors.

Alleles ; Asian Continental Ancestry Group ; genetics ; Gene Frequency ; Genotype ; HLA-C Antigens ; genetics ; Haplotypes ; Histocompatibility Testing ; Humans ; Ligands ; Polymorphism, Genetic ; Receptors, KIR2DL1 ; genetics

This study was aimed to investigate the biological characteristics of osteoblasts from patients with myelodysplastic syndrome (MDS) and their supportive capacity for hematopoiesis in vitro. A two-dimensional culture system was constructed by using osteoblasts derived from human marrow mesenchymal stem cells (MSC); MSCs were isolated from bone marrow of MDS patients and normal individuals and were cultured; the third passage of MSCs were induced into osteoblasts which were treated with mitomycin C and confluenced into a feeder layer. Ficolled bone marrow mononuclear cells were obtained from normal individuals and seeded into the two-dimensional culture system to culture in vitro without exogenous cytokines. By using colony-forming assay, the ability of the two-dimensional system to culture HPCs was observed. The cytokine expression of osteoblasts from MDS patient bone marrows in mRNA level was detected by RT-PCR and was compared with human osteoblast cell line hFOB1.19. The results showed that the osteoblasts from MDS patients could support short-term survival of GM-CFC in condition without exogenous cytokines, that is, osteoblasts played a crucial role in regulation of HPC growth. The results of RT-PCR clearly demonstrated that the osteoblast cell line hFOB1.19 expressed SCF, IL-6, SDF-1alpha, G-CSF and GM-CSF. The same expression patterns of above cytokines were also seen in osteoblasts derived from BM-MSCs of MDS patients and normal individuals, but these cells did not express GM-CSF. It is concluded that the biological characteristics of osteoblasts from bone marrow of MDS patients are generally not different from those of osteoblasts from normal bone marrow. Both of them can support GM -CFC to form colonies in vitro, it may be associated with expressing important related cytokines by osteoblasts.
Cytokines ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; metabolism ; Granulocyte-Macrophage Progenitor Cells ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Interleukin-6 ; metabolism ; Myelodysplastic Syndromes ; metabolism ; pathology ; Osteoblasts ; metabolism ; physiology ; RNA, Messenger ; metabolism ; Stem Cell Factor ; metabolism